Mechanism of Adenlylate Kinases

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The objective of this project is to study the structure-function relationships of the enzyme adenylate kinase (AK) by a combination of genetic, biochemical, bioorganic, and biophysical techniques. (1) Site-directed mutagenesis will be used to identify the residues involved in the binding of substrates in the ground state and in the transition state. The three existing models (derived from NMR, X-ray, and theoretical studies) will be critically tested. The goal is to obtain a revised or a new model. (2) The effects of point mutation on the conformation of AK will be evaluated in two stages. The first stage is to use difference protein NMR, difference circular dichroism, guanidine-HCl-induced denaturation, thermal stability, and computer graphics to assess possible structural perturbations in the mutant and the potential structural roles of the residues. The second stage is to use proton NMR to evaluate conformational perturbations in selected mutants more quantitatively. (3) The above model will be further tested and refined by "complementary substrate mutagenesis" using various substrate analogues. The goal is to confirm the specific interactions between the enzymic residues and the substrates from the substrate side.